1. Field of the Invention
The present invention relates generally to methods for diagnosing or monitoring Alzheimer's disease. More particularly, the present invention relates to measuring the amount of tau protein and/or the amount of β amyloid peptide (x-≧41) in patient fluid samples and using these amounts as a diagnostic indicator.
Alzheimer's disease (AD) is a degenerative brain disorder characterized clinically by progressive loss of memory, cognition, reasoning, judgment and emotional stability that gradually leads to profound mental deterioration and ultimately death. AD is a very common cause of progressive mental failure (dementia) in aged humans and is believed to represent the fourth most common medical cause of death in the United States. AD has been observed in all races and ethnic groups worldwide and presents a major present and future public health problem. The disease is currently estimated to affect about two to three million individuals in the United States alone. AD is at present incurable. No treatment that effectively prevents AD or reverses its symptoms or course is currently known.
The brains of individuals with AD exhibit characteristic lesions termed senile plaques, and neurofibrillary tangles. Large numbers of these lesions are generally found in several areas of the human brain important for memory and cognitive function in patients with AD. Smaller numbers of these lesions in a more restricted anatomical distribution are sometimes found in the brains of aged humans who do not have clinical AD. Senile plaques and amyloid angiopathy also characterize the brains of individuals beyond a certain age with Trisomy 21 (Down's Syndrome) and Hereditary Cerebral Hemorrhage with Amyloidosis of the Dutch-Type (HCHWAD). At present, a definitive diagnosis of AD usually requires observing the aforementioned lesions in the brain tissue of patients who have died with the disease or, rarely, in small biopsied samples of brain tissue taken during an invasive neurosurgical procedure. The principal chemical constituent of the senile plaques and vascular amyloid deposits (amyloid angiopathy) characteristic of AD and the other disorders mentioned above is an approximately 4.2 kilodalton (kD) protein of about 39-43 amino acids designated the amyloid-β peptide (Aβ) or sometimes βAP, AβP or β/A4. Aβ was first purified and a partial amino acid sequence reported in Glenner and Wong (1984) Biochem. Biophys. Res. Commun. 120:885-890. The isolation procedure and the sequence data for the first 28 amino acids are described in U.S. Pat. No. 4,666,829. Forms of Aβ having amino acids beyond number 40 were first reported by Kang et al. (1987) Nature 325:733-736.
Roher et al. (1993) Proc. Natl. Acad. Sci. USA 90:10836-840 showed that Aβ(1-42) is the major constituent in neuritic plaques (90%) with significant amounts of isomerized and racemized aspartyl residues. The authors also showed that Aβ(17-42) also predominates in diffuse plaques (70%), while Aβ(1-40) is the major constituent in the meningovascular plaques, comprising 60% of the total Aβ and, in parenchymal vessel deposits Aβ(1-42) represents 75% of the total Aβ. Iwatsubo et al. (1994) Neuron 13:45-53 showed that Aβ42(43)-positive senile plaques are the major species in sporadic AD brain.
Molecular biological and protein chemical analyses conducted during the last several years have shown that Aβ is a small fragment of a much larger precursor protein, referred to as the β-amyloid precursor protein (APP), that is normally produced by cells in many tissues of various animals, including humans. Knowledge of the structure of the gene encoding APP has demonstrated that Aβ arises as a peptide fragment that is cleaved from the carboxy-terminal end of APP by as-yet-unknown enzymes (proteases). The precise biochemical mechanism by which the Aβ fragment is cleaved from APP and subsequently deposited as amyloid plaques in the cerebral tissue and in the walls of cerebral and meningeal blood vessels is currently unknown.
Several lines of evidence indicate that progressive cerebral deposition of Aβ plays a seminal role in the pathogenesis of AD and can precede cognitive symptoms by years or decades (for review, see Selkoe (1994) J. Neuropath. and Exp. Neurol. 53:438-447 and Selkoe (1991) Neuron 6:487). The single most important line of evidence is the discovery in 1991 that missense DNA mutations at amino acid 717 of the 770-amino acid isoform of APP can be found in affected members but not unaffected members of several families with a genetically determined (familial) form of AD (Goate et al. (1991) Nature 349:704-706; Chartier Harlan et al. (1991) Nature 353:844-846; and Murrell et al. (1991) Science 254:97-99). Suzuki et al. (1994) Science 264:1336-1340 showed that in persons with the 717 mutation, there is a higher percentage of Aβ(−42) than Aβ(−40).
In addition, a double mutation changing lysine595-methionine596 to asparagine595-leucine596 (with reference to the 695 isoform) found in a Swedish family was reported in 1992 (Mullan et al. (1992) Nature Genet 1:345-347) and is referred to as the Swedish variant. Genetic linkage analyses have demonstrated that these mutations, as well as certain other mutations in the APP gene, are the specific molecular cause of AD in the affected members of such families. In addition, a mutation at amino acid 693 of the 770-amino acid isoform of APP has been identified as the cause of the Aβ deposition disease, HCHWA-D, and a change from alanine to glycine at amino acid 692 appears to cause a phenotype that resembles AD in some patients but HCHWA-D in others. The discovery of these and other mutations in APP in genetically based cases of AD argues that alteration of APP and subsequent deposition of its Aβ fragment can cause AD.
Neurofibrillary tangles are composed mainly of the microtubule protein, tau. Z. S. Khachaturian (1985) Arch. Neurol. 42:1097-1105. Recent studies have shown that tau is elevated in the CSF of Alzheimer's disease patients. M. Vandermeeren et al. (1993) J. Neurochem. 61:1828-1834.
Despite the progress which has been made in understanding the underlying mechanisms of AD, there remains a need to develop methods for use in diagnosis of the disease. While the level of tau is of some help in diagnosing Alzheimer's disease (M. Vandermeeren et al., supra) more markers, and more specific markers would be helpful. It would be further desirable to provide methods for use in diagnosis of Aβ-related conditions, where the diagnosis is based at least in part on detection of Aβ and related fragments in patient fluid samples. Specific assays for Aβ detection should be capable of detecting Aβ and related fragments in fluid samples at very low concentrations as well as distinguishing between Aβ and other fragments of APP which may be present in the sample.
2. Description of the Background Art
Glenner and Wong (1984) Biochem. Biophys. Res. Commun. 120:885-890 and U.S. Pat. No. 4,666,829, are discussed above. The '829 patent suggests the use of an antibody to the 28 amino acid Aβ fragment to detect “Alzheimer's Amyloid Polypeptide” in a patient sample and diagnose AD. No data demonstrating detection or diagnosis are presented.
Numerous biochemical electron microscopic and immunochemical studies have reported that Aβ is highly insoluble in physiologic solutions at normal pH. See, for example, Glenner and Wong (1984) Biochem. Biophys. Res. Commun. 122:1131-1135; Masters et al. (1985) Proc. Natl. Acad. Sci. USA 82:4245-4249; Selkoe et al. (1986) J. Neurochem. 46:1820-1834; Joachim et al. (1988) Brain Research 474:100-111; Hilbich et al. (1991) J. Mol. Biol. 218:149-163; Barrow and Zagorski (1991) Science 253:179-182; and Burdick et al. (1992) J. Biol. Chem. 267:546-554. Furthermore, this insolubility was predicted by and is consistent with the amino acid sequence of Aβ which includes a stretch of hydrophobic amino acids that constitutes part of the region that anchors the parent protein (APP) in the lipid membranes of cells. Hydrophobic, lipid-anchoring proteins such as Aβ are predicted to remain associated with cellular membranes or membrane fragments and thus not be present in physiologic extracellular fluids. The aforementioned studies and many others have reported the insolubility in physiologic solution of native Aβ purified from AD brain amyloid deposits or of synthetic peptides containing the Aβ sequence. The extraction of Aβ from cerebral amyloid deposits and its subsequent solubilization has required the use of strong, non-physiologic solvents and denaturants. Physiologic, buffered salt solutions that mimic the extracellular fluids of human tissues have uniformly failed to solubilize Aβ.
Separate attempts to detect APP or fragments thereof in plasma or CSF have also been undertaken. A large secreted fragment of APP that does not contain the intact Aβ region has been found in human cerebrospinal fluid (Palmert et al. (1989) Proc. Natl. Acad. Sci. USA 86:6338-6342; Weidemann et al. (1989) Cell 57:115-126; Henriksson et al. (1991) J. Neurochem. 56:1037-1042; Palmert et al. (1990) Neurology 40:1028-1034; and Seubert et al. (1993) Nature 361:260-263) and in plasma (Podlisny et al. (1990) Biochem. Biophys. Res. Commun. 167:1094-1101). The detection of fragments of the carboxy-terminal portion of APP in plasma has also been reported (Rumble et al. (1989) N. Engl. J. Med. 320:1446-1452), as has the failure to detect such fragments (Schlossmacher et al. (1992) Neurobiol. Aging 13:421-434).
Despite the apparent insolubility of native and synthetic Aβ, it had been speculated that Aβ might occur in body fluids, such as cerebrospinal fluid (CSF) or plasma (Wong et al. (1984) Proc. Natl. Acad. Sci. USA 92:8729-8732; Selkoe (1986) Neurobiol. Aging 7:425-432; Pardridge et al. (1987) Biochem. Biophys. Res. Commun. 145:241-248; Joachim et al. (1989) Nature 341:226-230; Selkoe et al. (1989) Neurobiol. Aging 10:387-395).
Several attempts to measure Aβ in CSF and plasma have been reported by both radioimmunoassay methods (WO90/12870 published Nov. 1, 1990) and sandwich ELISAs (Wisniewski in Alzheimer's Disease, eds. Becker and Giacobini, Taylor and Francas, N.Y. pg. 206, 1990; Kim and Wisniewski in Techniques in Diagnostic Pathology, eds. Bullock et al., Academic Press, Boston pg. 106; and WO90/12871 published Nov. 1, 1990). While these reports detected very low levels of Aβ immunoreactivity in bodily fluids, attempts to directly purify and characterize this immunoreactivity further and determine whether it represented Aβ were not pursued, and the efforts were abandoned. The possibility of Aβ production by cultured cells was neither considered nor demonstrated.
Retrospectively, the inability to readily detect Aβ in bodily fluids was likely due to the presence of amyloid precursor fragments with overlapping regions or fragments of Aβ that obscured measurements and to the lack of antibodies completely specific for intact Aβ. This is presumably because the antibodies used by both groups would cross-react with other APP fragments containing part of Aβ known to be present in CSF thereby interfering with the measurement, if any, of intact Aβ. These difficulties have been overcome with the use of monoclonal antibodies specific to an epitope in the central junction region of intact Aβ (Seubert et al. (1992) Nature 359:325-327).
Seubert et al. (1992) Nature 359:325-327 and Shoji et al. Science (1992) 258:126-129 provided the first biochemical evidence for the presence of discrete Aβ in bodily fluids. Vigo-Pelfrey et al. (1993) J. Neurochem. 61:1965-1968 reported the identification of many Aβ species in cerebrospinal fluid.